PROJECT SUMMARY/ABTRACT General anesthetics work in a concentration-dependent manner on the central nervous system (CNS) to induce loss of consciousness and block the experience of pain. Despite advances in our understanding of the molecular mechanisms of general anesthetics, how anesthetics alter CNS functioning to abolish the perception of pain is not well understood. A major impediment to progress has been a lack of understanding as to how general anesthetics exert their effects at the circuit level, particularly within regions of the cerebral cortex that process information relevant to the experience of pain. The processing of sensory stimuli by cortical circuits is tightly regulated by functionally distinct subpopulations of cortical neurons that have dissociable contributions in modulating local neural activity and information processing. However, how functionally distinct subpopulations of cortical neurons contribute to altered network activity during general anesthesia remains largely unexplored. This proposal aims to address two key questions: (1) Are functionally distinct neurons within the cortex (e.g., excitatory versus inhibitory, as well as molecularly distinct subclasses of inhibitory interneurons) differentially susceptible to general anesthetics? (Specific Aims 1 and 2) (2) Which subpopulations of functionally distinct neurons are activated by noxious stimuli, and are these responses altered during general anesthesia? (Specific Aim 3) Using miniature epifluorescent and two-photon calcium imaging to simultaneously monitor the in vivo activity of hundreds individual neurons within their larger cortical ensembles in the mouse during general anesthesia, this proposal aims to uncover the mechanisms through which a major volatile anesthetic, isoflurane, produces analgesia. Genetic approaches will be used to selectively fluorescently label functionally distinct subpopulations of cortical neurons. To determine whether functionally distinct populations of cortical neurons are differentially affected by isoflurane anesthesia, neural activity is monitored before, during and after anesthesia to compare the responses of (1) excitatory versus inhibitory neurons and (2) molecularly distinct subpopulations of inhibitory interneurons to the overall neuronal population. The responses of these distinct neuronal subpopulations will then be monitored during an acute, noxious stimulus to determine how isoflurane anesthesia interferes with cortical circuitry to produce analgesia. These studies are focused on two regions implicated in the generation of the pain percept: the anterior cingulate (ACC) and primary somatosensory (SI) cortices, which respectively process the affective and discriminative aspects of pain.